Ovarian carcinoma is the most common cause of death from a gynecological malignancy in the United States. Over ⅔ of the patients have advanced stage disease at presentation for which systemic chemotherapy is indicated after surgical debulking. Standard therapy consists of cisplatin or carboplatin with paclitaxel, and excellent response rates are observed; however, recurrence is common, and the majority of patients still die of disease progression. Ovarian cancer has a fairly unique natural history in humans. Even patients with advanced stages of the disease often have their disease confined to their abdomen for extended periods of time. The disease often stays localized to the abdomen and presents great difficulty for the patient by obstruction of the intestines or ureters. This natural history makes the possibility of locally-targeted therapy realistic. As a result, intraperitoneal therapies have been developed for the local administration of chemotherapeutic agents into the peritoneal cavity.
Gene therapy is among the experimental strategies for patients with cancer who have failed standard therapy. Despite more than 100 gene therapy trials, evidence of success is very limited. Roth, J. A., Cristiano, R. J., Roth, J. A. & Cristiano, R. J., Gene therapy for cancer: what have we done and where are we going? J. Natl. Cancer Inst. 89(1), 21–39 (1997). One strategy which has been explored for treating cancer is the artificial creation of differences between normal and neoplastic cells through prophylactic use of gene insertion techniques. In other words, manufacturing biochemical differences which can be exploited to systematically and specifically target neoplastic cells for destruction. Gene insertion protocols are used to artificially manufacture biochemical differences in target tumor cells which are then exploited to selectively kill these cells. One system which has received much attention to date is the Herpes Simplex Virus Ganciclovir System.
Transformation of tumor cells with the gene encoding Herpes Simplex Virus thymidine kinase (HSVtk) and subsequent treatment with anti-viral agents such as ganciclovir (GCV) has been previously accomplished and has proven to be operable in vivo in animals and humans. See Gene Therapy for the Treatment of Recurrent Pediatric Malignant Astrocytomas With In Vivo Tumor Transduction With Herpes Simplex Thymidine Kinase Gene/Ganciclovir System, Raffel, C. et al., Human Gene Therapy 5(7), p. 863–90, July 1994. The HSVtk gene is negative selectable marker or “suicide” gene of which most investigators in the field are well aware and versed in how the system is supposed to function. HSVtk sensitizes transduced tumor cells to GCV. Moolten, F. L., Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: Paradigm for a prospective cancer control strategy Cancer Res. 46, 5276–5281 (1986); Moolten, F. L. & Wells, J. M., Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors J. Nat. Cancer Inst. 82, 297–300 (1990); Moolten, F. L., Wells, J. M., Heyman, R. A. & Evans, R. M., Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene Human Gene Ther. 1, 125–134 (1990); Plautz, G., Nabel, E. G. & Nabel, G. J., Selective elimination of recombinant genes in vivo with a suicide retroviral vector New Biologist 3(7), 709–715 (1991). GCV is phosphorylated by HSVtk resulting in a monophosphate that cellular kinases convert to GCV-triphosphate which inhibits DNA replication and causes cell death. An interesting in vitro and in vivo observation with HSVtk is that only a portion of tumor cells need to be transduced with this gene to induce complete tumor destruction. This metabolic cooperation is a form of “bystander effect” and is due in large part to the transfer of phosphorylated GCV between cells through gap junctions. Moolten, F. L., Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: Paradigm for a prospective cancer control strategy Cancer Res. 46, 5276–5281 (1986); Culver, K. W., Ram, Z., Walbridge, S., Ishii, H., Oldfield, E. H., and Blaese, R. M. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors Science 256, 1550–1552 (1992); Nielsen, C. S., Moorman, D. W., Levy, J. P. & Link, C. J., Jr., Herpes simplex thymidine kinase gene transfer is required for complete regression of murine colon adenocarcinoma Am. Surg. 63(7), 617–620 (1997); Bi, W. L., Parysek, L. M., Warnick, R. & Stambrook, P. J., In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSVtk retroviral gene therapy Hum. Gene Ther. 4, 725–731 (1993); Freeman, S. M., Abboud, C. N., Whartenby, K. A., Packman, C. H., Koeplin, D. S., Moolten, F. L., and Abraham, G. N. The bystander effect: tumor regression when a fraction of the tumor mass is genetically modified Cancer Res. 53, 5274–5283 (1993); Ishii-Morita, H., Agbaria, R., Mullen, C. A., Hirano, H., Koeplin, D. A., Ram, Z., Oldfield, E. H., Johns, D. G., and Blaese, R. M. Mechanism of ‘bystander effect’ killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment Gene Ther. 4(3), 244–51 (1997); Link, C. J., Jr., Kolb, E. M. & Muldoon, R. R., Preliminary in vitro efficacy and toxicities studies of the herpes simplex thymidine kinase gene system for the treatment of breast cancer Hybridoma 14(2), 143–7 (1995); Samejima, Y. & Meruelo, D., ‘Bystander killing’ induces apoptosis and is inhibited by forskolin Gene Therapy 2, 50–58 (1995); Vrionis, F. D., Wu, J. K, Qi, P., Waltzman, M., Cherington, V., and Spray, D. C. The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HSVtk) gene is dependent on connexin expression and cell communication via gap junctions Gene Ther. 4(6), 577–85 (1997). The implantation of vector producer cells (VPC) to deliver genes, such as the HSVtk gene, into tumor cells was first demonstrated in a brain tumor model by engrafting lacZ VPC into rodents with C6 glioma tumors. Short, M. P., Choi, B. C., Lee, J. K., Malick, A., Breakefield, X. O., and Martuza, R. L. Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line J. Neurosci. Res. 27, 427–439 (1990). In these reported animal tumor models, all of the rodents are αgal+. Since these were αgal+ models, hyperacute rejection is not contributing to the observed responses with the HSVtk system. A human, however, is different.
Three prior human trials of murine HSVtk VPC have been reported; two for treatment of brain tumors and one for melanoma. Results from the first clinical trial conducted at the NCI using murine VPC xenografts were recently reported in Nature Medicine. Ram, Z., Culver, K. W., Oshiro, E. M., Viola, J., DeVroom, H. L., Otto, E., Long, Z., Chiang, Y., McGarrity, G. J., Muul, L. M., Katz, D., Blaese, R. M., and Oldfield, E. H. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells Nature Med. 3, 1354–1361 (1997). This trial used multiple stereotaxic injections to introduce murine HSVtk VPC into the enhancing portion of brain tumors. Antitumor activity was observed in selected local tumor deposits in 5 of 19 tumors injected in 13 patients. Ram, Z. et al., Nature Med. 3, 1354–1361 (1997). It is important to note that very limited (minimal) gene transfer was observed. The sterotaxic injections required multiple needle passages to try to distribute the VPC throughout the tumors resulting in severe hemorrhage that required surgery in 2 patients and MRI-visible bleeding in most of the other tumors. The authors theorized that the observed responses were secondary to the cell-to-cell transfer of phosphorylated GCV as “the major mechanism” of the bystander effect and concluded that “non-immune bystander mechanisms were critical.” Ram, Z. et al., Nature Med. 3, 1354–1361 (1997). They further stated that the observed responses were “probably not a result of immune mechanisms.” In light of data presented in this application and data from others demonstrating rapid destruction of murine VPC by human serum, these conclusions are unwarranted. Rollins, S. A., Birks, C. W., Setter, E., Squinto, S. P. & Rother, R. P., Retroviral vector producer cell killing in human serum is mediated by natural antibody and complement: Strategies for evading the humoral immune response Human Gene Therapy 7, 619–626 (1996); Link, C. J., Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D., in Cancer Gene Therapy (eds Mazarakis, H. & Swart, S. J.) 135–152 (IBC Library Series, London, United Kingdom, 1997). We suggest that the contrast enhancement reported along the needle tracts of VPC injections, as well as the transient volume increase of the tumors immediately after injection, was more likely to be the result of antibodies (Ab) and complement-mediated hyperacute rejection against the murine cells. Of note, the authors did report an increase in VPC binding Ab that peaked 21-142 days after VPC injection; these Ab may represent anti-αgal Ab. Ram, Z. et al., Nature Med. 3, 1354–1361 (1997). Other groups have shown that anti-pig Ab present in human serum are predominantly anti-αgal IgM Ab. Vaughan, H. A., McKenzie, I. F. & Sandrin, M. S. Biochemical studies of pig xenoantigens detected by naturally occurring human antibodies and the galactose alpha(1,3)galactose reactive lectin Transplantation 59(1), 102–9 (1995). The time frame of Ab titer increase is similar to that reported for diabetic patients transplanted with porcine islet xenografts and with our results with murine VPC intraperitoneal infusions discussed below. Galili, U., Tibell, A., Samuelsson, B., Rydberg, L. & Groth, C. G. Increased anti-Gal activity in diabetic patients transplanted with fetal porcine islet cell clusters Transplantation 59 (11), 1549–56 (1995).
A second brain tumor trial was recently reported by Klaztmann and colleagues. Klatzman, D., Valery, C. A., Bensimon, G., Marro, B., Boyer, O., Mokhtari, K., Diquet, B., Salzman, J.-L., Philippon, J., and Glioblastoma, S.G.o.G.T.f. A phase I/II dose escalation study of Herpes simplex virus type 1 thymidine kinase “suicide” gene therapy for recurrent glioblastoma Human Gene Ther. 9, 2595–2604 (1998). M11 murine VPC producing HSVtk retroviral vector were injected into the tumor margin after surgical debulking. Seven days later, patients were treated with GCV. Twelve patients were treated without side effects that the physicians attributed to the VPC or GCV. The authors claimed that the observed responses were secondary to gene therapy. One patient was still alive without evidence of progression by MRI at 2.8 years after the procedure. Eleven of twelve patients had died; nine from tumor progression, one from head trauma, and one from pulmonary embolus. To relate these observations to the gene therapy, the authors suggested several indirect lines of evidence. First, M11 cells could be recovered from surgical drain fluids 24 hours after cell injections. However, M11 VPC were recovered from only 3 out of 6 patients even at this short time point. Furthermore, no viable cells were recovered at later time points. Again, neurosurgical procedures with local tumor resections are operations with active bleeding and oozing into the tumor bed where the cells were injected. In fact, a blood-brain barrier that has been quoted by a number of groups to protect murine VPC (Ram, Z., J. Neurosurg. 79, 400–7 (1993)) is mechanically destroyed by the scalpel during major surgical debulking. Thus, M11 cells were most likely quickly lysed by the presence of human serum. The ability to recover a few viable M11 cells from the surgical drain 1 day after up to 9.8×106 cells/cm2 were injected is not compelling evidence that gene transfer is accounting for these observations. The second indirect evidence proposed was that GCV plasma levels were in a therapeutic range to kill HSVtk expressing cells. It is not understood how the presence of an adequate GCV level provides evidence of causation without HSVtk gene transfer data. No gene transfer into glioblastoma cells was reported. The imaging and pathologic data from this trial does suggest that patients who did not demonstrate MRI progression at 4 months after VPC injection do show some efficacy; however, the relationship between these observed responses and HSVtk and GCV is highly speculative.
One other study was performed on melanoma patients with non-CNS malignancy. Klatzman, D. et al., Human Gene Ther. 9, 2585–2594 (1998). Eight patients were treated by the direct injection of murine M11 packaging cells that produced HSVtk vector. The total cell dose ranged from 8×107 to 12.5×108 cells that were directly injected into tumors. Inflammatory reactions were common immediately after these xenogeneic VPC were injected. A rapid increase in tumor size was noted that peaked 24 hours later. The investigators attributed these effects to known pre-existing antibodies against xenogeneic antigens present on murine cells. This suggestion supports our findings. The very limited anti-tumor effects were some areas of local necrosis noted on biopsy samples. The lack of significant efficacy was attributed to poor gene transfer (<1% or none detected by PCR). Side effects of therapy consisted chiefly of local inflammatory reactions or fever when multiple injections were administered. This group suggested that murine VPC survival was enhanced by using intravenous immunoglobulin to delay xenogeneic hyperacute rejection. Gautreau, C., Kojima, T., Woimant, G., Cardoso, J., Devilier, P., and Houssin, D. Use of intravenous immunoglobulin to delay xenogeneic hyperacute rejection Transplantation 9, 903–907 (1995).
Hyperacute rejection of xenografts has been previously explored due to the great interest in using animals as a source of organs or tissues for humans. Strong immunological barriers to xenotransplants can destroy a transplanted solid organ within minutes, a process termed hyperacute rejection. This hyperacute rejection model of xenograft survival is typically a vascularized xenograft directly exposed to blood serum. Pruitt, S. K., Kirk, A. D., Bollinger, R. R., Marsh, J., Henry, C., Collins, B. H., Levin, J. L., Mault, J. R., Heinle, J. S., Ibrahim, S., Rudolph, A. R., Baldwin, I., William, M., and Sanfilippo, F. The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts Transplantation 57, 363–370 (1994). Research has demonstrated that hyperacute rejection with porcine xenografts transplanted into baboons occurs secondary to porcine α(1,3)galactosyltransferase [α(1,3)GT] gene expression and α(1,3)galactosyl epitopes (αgal) presentation. Pruitt, S. K. et al., Transplantation 57, 363–370 (1994); Platt, J. L., Vercellotti, G. M., Dalmasso, A. P., Matas, A. J., Bolman, R. M., Najarian, J. S., and Bach, F. H. transplantation of discordant xenografts: a review of progress Immunol. Today 17, 450–457 (1990). The enzyme α(1,3)GT is expressed in all mammalian species including Mus musculus, but not in Old World primates, apes, or humans. Galili, U. Shohet, S. B., Kobrin, E., Stults, C. L. & Macher, B. A., Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells J. Biol. Chem. 263(33), 17755–62 (1988). The α(1,3)GT gene is not active in humans due to the presence of two base pair frameshift mutations. Larsen, R. D., Rivera-Marrero, C. A., Ernst, L. K., Cummings, R. D. & Lowe, J. B., Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA J. Biol. Chem. 265(12), 7055–61 (1990). α(1,3)GT catalyzes the transfer of galactose from uridine diphosphate galactose (UDP-Gal) to the N-acetyl-lactosamine acceptors on carbohydrate side chains in a specific α(1,3)linkage on glycoproteins or glycolipids (Galβ1→4GlcNAc-R).
Galβ1→4GlcNAc-R+UDP-Gal→Galα1→3Galβ1→4GlcNAc-R Anti-αgal Ab present in the human serum can recognize this epitope. Galili, U. Shohet, S. B., Kobrin, E., Stults, C. L. & Macher, B. A., J. Biol. Chem. 263, 17755–62 (1988). In fact, pre-existing human Ab against αgal represent almost 1% of total human Ab (Galili, U., Evolution and pathophysiology of the human natural anti-y-galactosyl IgG (anti-Gal) antibody Springer Semin. Immunopathol. 15, 155–171 (1993)) and are the basis for complement-mediated hyperacute rejection. Sandrin, M. S., Vaughan, H. A., Dabkowski, P. L. & McKenzie, I. F. Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1–3)Gal epitopes Proc. Natl. Acad. Sci. U.S.A. 90(23), 11391–5 (1993). Human anti-αgal Ab are thought to arise in response to αgal structures on the surface of normal GI flora. The translocation of viable bacteria from the enteric lumen to the mesenteric lymph nodes is thought to stimulate the host immune response. Neonatal humans and baboons compared to their respective adults have very low titers of anti-αgal IgM Ab, the isotype most effective in binding complement. Xu, H., Edwards, N. M., Dong, X. & Michler, R. E. Age-related development of human preformed anti-porcine endothelial cell xenoantibody J. Thorac. Cardiovasc. Surg. 15, 1023 (1995); Minanov, O. P., Itescu, S., Neethling, F. A., Morgenthau, A. S., Kwiatkowski, P., and Cooper, D. K. Anti-gal IgG antibodies in sera of newborn humans and baboons and its significance pig xenotransplantation Transplantation 63, 182 (1997). Neonatal circulating xenoreactive Ab are of the IgG isotype, presumably attained by placental transfer of maternal IgG. Xu, H., Edwards, N. M., Dong, X. & Michler, R. E., J. Thorac. Cardiovasc. Surg. 15, 1023 (1995); Minanov, O. P. et al., Transplantation 63, 182 (1997); Galili, U., Springer Semin. Immunopathol. 15, 155 (1993). Since newborn gut is sterile, there is a time period in which newborn primates are not exposed to bacterial αgal moieties and, thus, lack de novo Ab directed against these epitopes.
Murine vector producing cells implanted into humans is another type of xenograft. It has been demonstrated that murine retroviral VPC and the viral vectors they produce express αgal and, therefore, are lysed by Ab and complement within 30 minutes after being exposed to human serum. Link, C. J., Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D., Cancer Gene Therapy (eds Mazarakis, H. & Swart, S. J.) 135–152 (IBC Library Series, London, United Kingdom, 1997); Welsh, R. M., Cooper, N. R., Jensen, F. C. & Oldstone, M. B., Human serum lyses RNA tumor viruses Nature 257, 612–614 (1975); Rother, R. P., Fodor, W. L., Springhorn, J. P., Birks, C. W., Setter, E., Sandrin, M. S., Squinto, S. P., and Rollins, S. A. A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody J. Exp. Med. 182(5), 1345–55 (1995). The effect of this serum inactivation on VPC and retroviruses is due to αgal expression on the cells. Link, C. J., Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D., Cancer Gene Therapy (eds Mazarakis, H. & Swart, S. J.) 135–152 (IBC Library Series, London, United Kingdom, 1997); Rother, R. P. et al., J. Exp. Med. 182, 1345–55 (1995); Rother, R. P., Squinto, S. P., Mason, J. M. & Rollins, S. A., Protection of retroviral vector particles in human blood through complement inhibition Hum. Gene Ther. 6(4), 429–35 (1995).
Viral vectors can efficiently transduce human tumor cells with anti-tumor therapeutic genes. As part of a study of blocking retroviral destruction by using human packaging cells, the transfer of the porcine α(1,3)GT to human fibroblasts was shown to result in sensitivity to Ab and complement destruction. Collins, M. K., Takeuchi, Y., Cosset, F. L., Tailor, C. & Weiss, R. A. Development of recombinant retroviruses suitable for in vivo gene delivery Cold Spring Harbor Meeting: Gene Therapy September 1994, 97 (1994). We have found that the retroviral transduction of human tumor cells with the α(1,3)GT gene resulted in its expression. These human cells displayed αgal and became sensitive to human serum. In this project, murine VPC are employed to deliver retroviral vector to intraperitoneal ovarian tumors. We are interested in the biology of glycosylation and the immunologic effect of αgal epitopes. Therefore, the project goal is to further understand mechanisms of hyperacute rejection. Hyperacute rejection may cause a strong intraperitoneal inflammatory response, that through an innocent bystander mechanism, destroys ovarian cancer cells. The process of local tumor destruction might result in the disruption of tumor anergy. This has been a common strategy in gene modification trials. Previous attempts of immunotherapy have mainly employed single cytokine molecules (e.g., IL-2, GM-CSF) (Golumbek, P. T., Lazenby, A. J., Levitskky, H. I., Jaffee, L. M., Karasuyama, H., Baker, M., and Pardoll, D. M. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4 Science 254, 713716 (1991); Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R. Vaccination with irradiated tumor cells engineered to secrete murine GM-CSF stimulates potent, specific, and long lasting anti-tumor immunity Proc. Natl. Acad. Sci. (USA) 90, 3539–3543 (1993); Fearon, E. R., Pardoll, D. M., Itaya, T., Golumbek, P., Levitsky, H. I., Simons, J. W., Karasuyama, H., Vogelstein, B., and Frost, P. Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response Cell 60, 397–403 (1990)) or single rejection antigens (e.g., HLA B7, melanoma specific tumor antigen). Nabel, G. J., Nabel, E., Yang, Z., Fox, B. A., Plautz, G. E., Gao, X., Huang, L., Shu, S., Gordon, D., and Chang, A. E. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity, and lack of toxicity in humans Proc. Natl. Acad. Sci. (USA) 90, 11307–11311 (1993); Reeves, M. E., Royal, R. E., Lam, J. S., Rosenberg, S. A. & Hwu, P. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene Cancer Res. 56(24), 5672–7 (1996).
Since anti-tumor gene therapy requires highly efficient gene transfer and expression of therapeutic genes (Roth, J. A., Cristiano, R. J., Roth, J. A. & Cristiano, R. J. Gene therapy for cancer: what we have done and where are we going? J. Natl. Cancer Inst. 89(1), 21–39 (1997)) and the Goldie-Coldman hypothesis predicts that spontaneous mutations in cancer cells provide resistance to chemotherapy and that therapeutic failures are directly related to tumor burden (Goldie, J. H. & Coldman, A. J. A mathematic model for relating the drug sensitivity of tumors to their spontaneous mutation rate Cancer Treat. Rep. 63(11–12), 1727–33 (1979)), multiple, independent therapeutic targets need to be attacked for success. The predicted tumor resistance will likely extend to gene therapy as well; for example, our group has demonstrated that resistance to HSVtk gene and GCV killing is common in tumor cells. Seregina, T., Levy, J. & Link, C. in Fourth International Conference on the Gene Therapy of Cancer Vol. 2 (eds Sobol, R. E. & Royston, I.) A332 (Appleton & Lange, San Diego, Calif., 1995). In the future, gene delivery methods that transfer multiple therapeutic genes in concert or genes with multiple mechanisms of action will dominate approaches for cancer treatment.
All references cited throughout this application are hereby incorporated by reference.
Based on the foregoing, it is desirable that a less complicated system without use of vectors be available. Using VPC could be eliminated using the present invention, thus, taking away the additional risk to the patient of using viral vectors. The present invention is able to create antitumor responses without the need for gene transfection and addition of a prodrug.